The invention relates to devices and methods for analyzing cellular growth in vitro.
Tumors grow through two primary processes: proliferation and invasion. Proliferative growth represents the increase in size of the central tumor mass through the division of cells. Invasive growth occurs in tissues in the regions adjacent to and around the central tumor mass. In the invasion process, individual tumor cells detach from the central tumor mass and begin to actively move through the surrounding, non-tumorous tissue, either by compression or enzymatic degradation. The presumably highly branched cells formed by these invading cells represent a dynamically evolving network pattern. The chains formed in the invasive network can be as thin as a single cell in width. Further, the invading cells are significantly elongated along the direction in which they are traveling.
Malignant tumors such as highly malignant brain tumors (e.g., gliomas and glioblastoma multiform) have several features such as proliferation, invasion, central necrosis/apoptosis and neo-vascularization. Tumors outside the central nervous system show extensive metastasis by way of the blood circulatory and lymphatic systems.
Several in vitro assays have been described that are designed to measure either cell proliferation, migration, or invasion. For example, a cell colony/spheroid-agarose assay uses cell suspensions within, or multicellular tumor spheroids placed on top of agarose (in a cell culture dish), which is then covered with cell culture medium. Carlsson J., Int. J. Cancer, 20:129-136, 1977. The assay is designed to study the growth dynamics of the cell colonies or spheroid. The medium superlayer distributes growth factors and growth limiting factors produced by the tumor, without preserving their important regional concentration differences (i.e., higher concentration around the tumor). Also, the superlayer has to be changed routinely, altering the environmental setting.
A 2D migration assay can be used to describe the movement of a cell population from a central area in an expanding circle. Giese A., Neurosurgery, 37:294-302, 1995. The cells are placed in a cell culture dish and covered with medium. Aside from the edge of the dish, there is no mechanical confinement and no chemo-gradients can be established due to xe2x80x9cequalizingxe2x80x9d in the medium superlayer.
Invasiveness assays (e.g., commercially available through Costar(copyright) as the 24-well Transwell(trademark) System) use a medium-supplemented, two-chamber system, which is designed to detect cell migration between the two chambers. Repesh L. A., Invasion Metastasis, 9:192-208, 1989. The insert (top chamber) has a polycarbonate membrane on the bottom, which has a number of pores (8 xcexcm size). After a certain number of cells are placed onto this membrane, they start to move through the pores and drop into the lower chamber where they either start anchorage-independent growth or eventually attach. After an observation period the cell number in both chambers is counted (Coulter Counter System(copyright)) and a ratio indicates the specific invasive potential of the cell line used. A variation of the assay uses a Matrigel(copyright) layer on top of the polycarbonate membrane to investigate the enzymatic activity of the cells to digest their way towards the pores.
A spheroid-fetal rat brain aggregate assay uses rat brain aggregates co-cultured on a medium/agar-layer and covered with a cell-culture medium that is changed routinely. Khoshyomn S., J. Neuro-Oncology, 38:1-10, 1998. The migration capacity of the tumor cells is determined by the destruction of the rat brain aggregate, not by the direct measurement of cell branches.
The invention is based on the discovery that cells can be grown in vitro in an enclosed device that allows for a three-dimensional measurement of both their proliferative and invasive properties. By growing the cells in an enclosed matrix that resembles the environment the cells confront in vivo, the cells can divide, invade, and form branched networks as they are thought to do in living tissue, e.g., in an individual. Propagating the cells in vitro in this manner allows for the imaging and temporal-spatial analysis of cells and cellular behavior that cannot be easily achieved when the cells are grown inside an organism. The methods and devices of the invention are particularly useful for studying the growth of tumor cells in vitro. The assay devices of the invention can thus be used as model systems to study cancer biology and to evaluate the efficacy of anti-cancer therapeutics.
In general, the invention features an assay device for measuring the proliferation and/or invasion of cells, e.g., tumor cells. The device includes a test chamber and a first delivery chamber arranged to contact the test chamber. The device can also include a control chamber, e.g., arranged to contact the first delivery chamber or a second delivery chamber. The first delivery chamber includes a wall with an opening to enable fluid communication between the first delivery chamber and the test chamber. The device also includes a hollow cylinder enclosing a lumen and arranged within the first delivery chamber, the cylinder including a wall with a hole that can be aligned with the opening in the first delivery chamber wall to enable fluid communication between the cylinder lumen and the test chamber.
The assay device can further include a cover that sealingly contacts the delivery chamber, the test chamber, and the control chamber, if present. The assay device can also include a moveable interior wall that is arranged within the test chamber to be moved laterally within the test chamber, e.g., by turning screws located in holes in an outer wall of the test chamber. The assay device optionally includes a second moveable interior wall that is arranged within the control chamber to be moved laterally within the control chamber, e.g., by turning screws located in holes in an outer wall of the control chamber.
The invention also features an assay device that includes a plurality of cylinders, each having a hole that can be aligned with the opening in the delivery chamber wall to enable fluid communication between the cylinder and the test chamber, wherein the cylinders are interchangeable and each has a hole of a different size. In addition, the assay device can also include a second delivery chamber arranged to contact the control chamber, e.g., in the same manner that the first delivery chamber is arranged to contact the test chamber. This would allow the control chamber to be exposed to a control fluid, as compared to a test fluid in the test chamber. The control chamber can include a moveable wall that is arranged to move within the control chamber.
The test chamber of an assay device can include an outer wall with an opening to enable fluid communication between the test chamber and the exterior of the assay device. An assay device can also include a hollow insert constructed to fit within the test chamber. The hollow insert can contain a moveable wall that is arranged to move within the insert.
In another embodiment, the invention features an assay system for measuring the proliferation and/or invasion of cells. The assay system includes an assay device of the invention, a pump having an input and an output, a first conduit that connects one end of the cylinder to the pump input, and a second conduit that connects a second end of the cylinder to the pump output to permit flow of fluid, e.g., a liquid or gas, from the pump, through the cylinder in the delivery chamber of the device, and back to the pump.
The assay system can also include an injection port connected to a conduit that permits the introduction of substances into the system, e.g., by microinjection. Additionally, the assay system can include a device of the invention that includes a first moveable interior wall that is arranged within the test chamber to be moved laterally within the test chamber, e.g., by turning screws located in holes in an outer wall of the test chamber. Furthermore, the assay system can include a device of the invention including a second moveable interior wall that is arranged within a control chamber to be moved laterally within the control chamber, e.g., by turning screws located in holes in an outer wall of the control chamber. The advancement of the walls allows for a continuous controlled increase of the mechanical confinement within the chambers and the study of its structural and functional impact on the distinct features of the cell system (e.g., proliferation and/or invasion).
The test chamber of an assay device of an assay system can include an outer wall with an opening to enable fluid communication between the test chamber and the exterior of the assay device. An assay system can also include a hollow insert constructed to fit within the test chamber. The hollow insert can contain a moveable wall that is arranged to move within the insert.
In another aspect, the invention features a method for detecting the proliferation of cells, e.g., tumor cells. This method includes the following steps: (1) placing one or more cells in a matrix within the test chamber of a device of the invention; (2) placing the device under conditions that permit the growth of the cells contained therein; (3) aligning the hole in the wall of the cylinder and the opening in the wall of the first delivery chamber to enable liquid medium to flow into the test chamber; (4) flowing liquid medium through the cylinder within the delivery chamber of the device; and (5) evaluating the proliferation of the cells within both the test chamber. The test chamber can include a moveable wall and the method can include moving the moveable wall. In one example, the placing of the one or more cells in a matrix within the test chamber includes placing the one or more cells within a matrix in a hollow insert, and placing the hollow insert within the test chamber. The insert can include a moveable wall and the method can include moving the moveable wall.
The method can also include flowing a gas through the cylinder within the delivery chamber of a device of the invention. The method can evaluate the proliferation of the tumor cells by counting the numbers of cells within the control and test chambers, e.g., by microscopic analysis of cells within the device. The tumor cells can optionally be extracted from the device for analysis.
The invention also features methods for detecting the proliferation of tumor cells wherein therapeutic agents are in the liquid medium that flows into the test chamber of the device. Additionally, according to the methods of the invention, therapeutic agents can be delivered to the test chamber of the device through holes in an outer wall of the test chamber. The methods also include the step of flowing liquid medium from the test chamber through a hole in the outer wall of the test chamber to the exterior of the device.
In another embodiment, the invention features a method for detecting invasion of tumor cells. The method includes the following steps: (1) placing tumor cells in a matrix within both the test chamber of a device of the invention; (2) placing the device under conditions that permit the invasion of the cells contained therein; (3) aligning the hole in the wall of the cylinder and the opening in the wall of the delivery chamber to enable liquid medium to flow into the test chamber; (4) flowing liquid medium through the cylinder within the delivery chamber of the device; and (5) evaluating invasion of the cells within the test chamber. The method also includes flowing a gas through the cylinder within the delivery chamber of the device. The method can evaluate the invasion of tumor cells by microscopic analysis of cells within the device. The tumor cells can be extracted from the device for analysis as part of the method.
The invention also features methods for detecting invasion of tumor cells, wherein therapeutic agents are included in the liquid medium that flows into the test chamber of the device. Additionally, according to the methods of the invention, therapeutic agents can be delivered to the test chamber of the device through holes in an outer wall of the test chamber.
The assay devices of the invention allow for dynamic three-dimensional measurements of the proliferation and invasion of multicellular systems such as tumor cells in an in vitro assay. The lack of a medium superlayer allows for some of the devices to be tilted to any angle and even flipped 180 degrees for true three-dimensional measurement, without an alteration of the internal environment. The new assay devices of the invention have the advantage of being able to investigate tumors as multi-featured systems.
The invention also provides advantages over in vivo assays. Single cell invasion cannot be easily studied in situ or in vivo (e.g., in animal models) because of the limitations of the resolution threshold of imaging methods. The new assays allow for in vitro growth of cells, e.g., tumor cells, in three dimensions over time so as to mimic tumor growth in vivo, but also render the cells accessible to a wide range of in vitro techniques that allow the study of their biology and their reaction to various agents, including potential therapeutics. The new assay systems allow for a continuous flow through gel-embedded cells and thus allows for repeated measurements of cells as they grow in a three-dimensional environment. The devices described herein allow for easier focusing on a tumor using microscopic techniques, thus increasing the ability to achieve sharp pictures from all angles. The advanced, new in vitro models also limit the amount of necessary in vivo experimentation by providing a pre-evaluation and a focus on promising drugs.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification will control. In addition, the described materials and methods are illustrative only and are not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and the claims.